Compositions and assays utilizing ADP or phosphate for detecting protein modulators

ABSTRACT

Described herein are methods which identify candidate agents as binding to a protein or as a modulator of the binding characteristics or biological activity of a protein. Generally, the methods involve the use of ADP or phosphate. The assays can be used in a high throughput system to obviate the cumbersome steps of using gels or radioactive materials.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/856,580, filed May 28, 2004, which is a continuation of U.S.application Ser. No. 10/106,665, filed Mar. 25, 2002, now U.S. Pat. No.6,743,599, which is a continuation of U.S. application Ser. No.09/724,990, filed Nov. 28, 2000, abandoned, which is a division of U.S.application Ser. No. 09/314,464, filed May 18, 1999, now U.S. Pat. No.6,410,254, the disclosure of which is incorporated herein by reference.

FIELD OF INVENTION

This invention is related to the use of adenosine diphosphate (ADP) orphosphate in assays for identifying compounds which bind to or modulatethe binding characteristics or biological activity of a protein.

BACKGROUND OF THE INVENTION

Drugs and other compounds intended for use in the diagnosis, cure,mitigation, treatment or prevention of disease in man or other animal orfor use in the agricultural arena, have made a significant impact on thepractice of modern medicine and on the agricultural arena. In somecases, such as in the development of vaccines, drugs have essentiallyeradicated once untreatable diseases. In the case of the agriculture,compounds have been developed which both extend the life and/or volumeof produce as well as kill unwanted plants where desirable. Therefore,the development of these compounds is of great interest.

Many useful compounds modulate the physical interaction of proteins.Traditionally, these protein—protein interactions have been evaluatedusing biochemical techniques, including chemical cross-linking,co-immunoprecipitation, co-fractionation and co-purification. Recentlygenetic systems have been invented to detect protein—proteininteractions. The first work was done in yeast systems, and was termedthe “yeast two-hybrid” system. The basic system requires aprotein—protein interaction in order to turn on transcription of areporter gene. Subsequent work was done in mammalian cells. See Fieldset al., Nature 340:245 (1989); Vasavada et al., PNAS USA 88:10686(1991); Fearon et al., PNAS USA 89:7958 (1992); Dang et al., Mol. Cell.Biol. 11:954 (1991); Chien et al., PNAS USA 88:9578 (1991); and U.S.Pat. Nos. 5,283,173, 5,667,973, 5,468,614, 5,525,490, and 5,637,463.

In another approach to drug discovery, studies are designed to determinethe biological activity of a protein. For example, the conditions suchas the specific substrate or stimulator required for an enzymaticreaction are investigated. Moreover, there are a number of studiesdesigned specifically for aide in the detection step in these assays.For example, one study discloses a spectrophotometric assay forinorganic phosphate (Pi) to probe the kinetics of Pi release frombiological systems such as GTPases and ATPases. Webb, PNAS, 89:4884–4887(1992). Another study reports on an enzymatic assay of inorganicphosphate in serum using nucleoside phosphorylase and xanthine oxidase.Ungerer, et al., Elsevier Clinica Chimica Act, 223:149–157 (1993). Acontinuous spectrophotometric assay for aspartate transcarbamylase andATPases is reported on in Rieger, et al., Anal. Biochem., 246:86–95(1997). There is also a study which reports on the measurement ofinorganic phosphate release using fluorescent probes and its applicationto actomysin subfragment 1 ATPase. Brune, et al., Biochem., 33:8262–8271(1994). U.S. Pat. No. 4,923,796 discloses a method for quantitativeenzymatic determination of ADP. Microtubule-stimulated adenosinetriphosphate (ATP) hydrolysis by kinesin is discussed in Hackney, J.Biol. Chem., 269(23):16508–16511 (1994). Furthermore, enzymaticfluorimetry and fluorimetric assays for ATPase activity are reported onin Greengard, Nature, 178:632–634 (1956) and Utpal and Siddhrtha,Biochem. J., 266:611–614 (1990), respectively.

In a different approach, modulators of an enzymatic reaction areinvestigated, wherein the conditions which allow the enzymatic reactionto occur are already known. For example, U.S. Pat. No. 5,759,795discloses an assay for identifying an inhibitor of a Hepatitis C VirusNS3 protein ATPase which involves a luciferase reaction. Luciferasereactions are known in the art. In the case of an ATPase inhibitor, thepresence of an ATPase inhibitor is indicated when ATP is available todrive the oxidation of luciferon by luciferase. This approach requiresATP but does not re-generate ATP.

Thus, while efforts have been made toward drug discovery, more efficientmeans are desirable. In particular, there is a need for an efficientsystem which can distinguish between a compound directly binding to asecond component, or whether the compound modulates the binding betweentwo other components, or whether the compound modulates the biologicalactivity of a known enzymatic reaction. Accordingly, it is an object ofthe present invention to provide methods of identifying compounds whicheither bind to or which modulate the binding characteristics or thebiological activity of a target protein. It is also an object to providecompositions for use in the assays provided herein.

SUMMARY OF THE INVENTION

The present invention provides methods which identify candidate agentsthat bind to a a protein or act as a modulator of the bindingcharacteristics or biological activity of a protein. In one embodiment,the method is performed in plurality simultaneously. For example, themethod can be performed at the same time on multiple assay mixtures in amulti-well screening plate as further described below. Furthermore, in apreferred embodiment, fluorescence or absorbance readouts are utilizedto determine enzymatic activity. Thus, in one aspect, the inventionprovides a high throughput screening system.

In one embodiment, the present invention provides a method ofidentifying a candidate agent as a modulator of the activity of a targetprotein. The method comprises adding a candidate agent to a mixturecomprising a target protein which directly or indirectly produces ADP orphosphate under conditions which normally allow the production of ADP orphosphate. The method further comprises subjecting the mixture to anenzymatic reaction which uses said ADP or phosphate as a substrate underconditions which normally allow the ADP or phosphate to be utilized anddetermining the level of activity of the enzymatic reaction. A change inthe level between the presence and absence of the candidate agentindicates a modulator of the target protein.

In one aspect, the target protein indirectly produces the ADP orphosphate by producing a substrate for a reaction which produces the ADPor phosphate. In another aspect, the target protein indirectly producesphosphate or ADP or phosphate by regulating an enzyme which produces ADPor phosphate. In yet a further aspect, the target protein directlyproduces phosphate or ADP.

In another aspect, the invention provides a method of identifying acandidate agent as a modulator of the activity of a target proteinwherein the target protein uses ADP or phosphate directly or indirectly.The method comprises adding a candidate agent to a mixture comprisingthe target protein under conditions which normally allow the utilizationof ADP or phosphate. The method further comprises determining the levelof utilization wherein a change in the level between the presence andabsence of the candidate agent indicates a modulator of the targetprotein.

In another embodiment provided herein is a method for identifyingwhether any two target proteins interact. The method comprises providinga first target chimera comprising a functional molecular motor bindingdomain and a first target protein. The method further comprisesproviding a second target chimera comprising a functional microtubulestimulated ATPase domain and a second target protein. Additionally, themethod comprises combining the first and second target chimeras underconditions which normally allow activity of a motor protein whichcomprises a molecular motor binding domain and a microtubule stimulatedATPase domain, wherein an increase in motor protein activity indicatesinteraction between the two target proteins.

In a further embodiment a method is provided for identifying whether acandidate agent is a modulator of at least one of any two targetproteins. The method comprises providing a first target chimeracomprising a functional molecular motor binding domain and a firsttarget protein and further providing a second target chimera comprisinga functional microtubule stimulated ATPase domain and a second targetprotein. Additionally, the method comprises combining the first andsecond target chimeras in the presence and absence of a candidate,wherein a change in motor protein activity, which requires both amolecular motor binding domain and a microtubule stimulated ATPase,between the presence and absence of a candidate agent indicates thecandidate agent is a modulator of at least one of the target proteins.

Additionally, provided herein is a chimeric protein comprising afunctional molecular motor binding domain and a target binding domainwherein the chimeric protein is independent of a functional microtubulestimulated ATPase domain. Also provided herein is a chimeric proteincomprising a functional microtubule stimulated ATPase domain and atarget binding domain, wherein the chimeric protein is independent of afunctional molecular motor binding domain.

In one aspect, a nucleic acid comprising a nucleic acid encoding achimeric protein in accordance with the present invention is provided.In another aspect a cell comprising a nucleic acid or a chimeric proteinin accordance with the present invention is provided.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides methods for the identification ofcandidate agents that bind to a target protein or serve as modulators ofthe biological activity of a target protein. These assays utilizevarious methods to measure, in ways amenable to high throughputscreening, the generation or consumption of ADP or phosphate. That is,target proteins that either directly or indirectly produce or consumeADP or phosphate may be screened in the present invention. Thus, byproviding assay systems that rapidly, efficiently and inexpensivelyassay ADP or phosphate, modulators (including both antagonists andagonists) of any test protein that directly or indirectly produces ADPor phosphate may be found. The present invention thus utilizes highthroughput assays that obviate the traditional cumbersome steps of usinggels or radioactive materials.

Accordingly, the present invention provides methods of screening oftarget proteins. By “target protein” herein is meant a protein thatdirectly or indirectly produces ADP or phosphate. The target proteinscan be from eukaryotes or procaryotes, including mammals, fungi,bacteria, insects, and plants, as well as viruses. In a preferredembodiment, the target proteins are from mammalian cells, with rodents(mice, rats, hamsters, guinea pigs and gerbils being preferred),primates and humans being preferred, and humans being particularlypreferred.

“Protein” in this context means a compound that comprises at least twocovalently attached amino acids and includes proteins, polypeptides,oligopeptides and peptides. The proteins may be made up of naturallyoccurring amino acids and peptide bonds, or synthetic peptidomimeticstructures. Thus “amino acid”, or “peptide residue”, as used hereinmeans both naturally occurring and synthetic amino acids. For example,homo-phenylalanine, citrulline and noreleucine are considered aminoacids for the purposes of the invention. “Amino acid” also includesimino acid residues such as proline and hydroxyproline. The side chainsmay be in either the (R) or the (S) configuration. In the preferredembodiment, the amino acids are in the (S) or L-configuration. Ifnon-naturally occurring side chains are used, non-amino acidsubstituents may be used, for example to prevent or retard in vivodegradations.

Suitable target proteins, include, but are not limited to, cytoskeletalproteins including, but not limited to, kinesins, myosins, tubulins,actins, tropomyosins, and troponins, with human proteins beingpreferred.

In a preferred embodiment, the target protein is a kinesin, includingmitotic kinesins. Mitotic kinesins are enzymes essential for assemblyand function of the mitotic spindle, but are not generally part of othermicrotubule structures, such as nerve processes. Mitotic kinesins playessential roles during all phases of mitosis. These enzymes are“molecular motors” that translate energy released by hydrolysis of ATPinto mechanical force which drives the directional movement of cellularcargoes along microtubules. The catalytic domain sufficient for thistask is a compact structure of approximately 340 amino acids. Duringmitosis, kinesins organize microtubules into the bipolar structure thatis the mitotic spindle. Kinesins mediate movement of chromosomes alongspindle microtubules, as well as structural changes in the mitoticspindle associated with specific phases of mitosis. Experimentalperturbation of mitotic kinesin function causes malformation ordysfunction of the mitotic spindle, frequently resulting in cell cyclearrest. From both the biological and enzymatic perspectives, theseenzymes are attractive targets for the discovery and development ofnovel anti-mitotic chemotherapeutics.

Suitable kinesins include, but are not limited to, Kin2, chromokinesin,Kif1A, KSP, CENP-E, MCAK, HSET and Kif15 is provided. K335, Q475, D679,FL1, P166, H195, FL2, E433, R494, E658, L360, K491, S553, M329, T340,S405, V465, T488, M1, M2, M3, M4, M5, M6, FL3, A2N370, A2M511, K519,E152.2, Q151.2, Q353, M472 and MKLP1. It is understood that unless aparticular species is named, the term “kinesin” includes homologsthereof which may have different nomenclature among species. Forexample, the human homolog of Kif1A is termed ATSV, the human homologueof Xenopus Eg5 is termed KSP, and human HSET corresponds to Chinesehamster CHO2.

By “kinesin protein activity” or grammatical equivalents herein is meantone of kinesin protein's biological activities, including, but notlimited to, its ability to affect ATP hydrolyzation. Other activitiesinclude microtubule binding, gliding, polymerazation/depolymerazation(effects on microtubule dynamics), binding to other proteins of thespindle, binding to proteins involved in cell-cycle control, or servingas a substrate to other enzymes, such as kinases or proteases andspecific kinesin cellular activities such as chromosome congregation,axonal transport, etc.

Methods of performing motility assays are well known to those of skillin the art (see, e.g., Hall, et al. (1996), Biophys. J., 71: 3467–3476,Turner et al., 1996, Anal. Biochem. 242 (1):20–5; Gittes et al., 1996,Biophys. J. 70(1): 418–29; Shirakawa et al., 1995, J. Exp. Biol. 198:1809–15; Winkelmann et al., 1995, Biophys. J. 68: 2444–53; Winkelmann etal., 1995, Biophys. J. 68: 72S, and the like).

In a preferred embodiment, the target protein directly or indirectlyproduces ADP and/or phosphate. Included in the definition of adenosinediphosphate (ADP) are ADP analogs, including, but not limited to,deoxyadenosine diphosphate (dADP) and adenosine analogs. As used herein,phosphate is used interchangeably with inorganic phosphate.

In a preferred embodiment, the target protein directly produces ADP orphosphate. In a preferred embodiment, the target protein is an enzymehaving activity which produces ADP and/or phosphate as a reactionproduct. For example, proteins which directly produce ADP include butare not limited to ATPases, kinases, GTPases, phosphatases andphosphorylases. Suitable ATPases include, but are not limited to,myosins, kinesins, dyneins, DNA gyrase, DNA helicase, topoisomerase Iand II, Na+-K+ ATPase, Ca2+ ATPase, F1 subunit of ATP synthase,terminase/DNA packaging protein; recA, heat shock proteins, NSF,katanin, SecA, 5-lipoxygenase, and actin. Suitable kinases include, butare not limited to, tyrosine kinases; serine-threonine kinases; receptortyrosine kinases; growth factor receptors including but not limited toinsulin receptor, epidermal growth factor receptor, platelet derivedgrowth factor receptor and fibroblast growth factor receptor; ErbB2;calmodulin dependent protein kinases; protein kinase A; protein kinaseC; myosin light chain kinase; CDK2 kinase; ROCK1 kinases; Src kinases;phosphorylase kinase; CheA; adenylate kinase; glycolytic kinases; EIF-2alpha protein kinases; and Abl. Suitable GTPases include, but are notlimited to, G proteins, Rho family GTPases: cdc42, RalA, RhoA and Rac1;Ras proteins; elongation factors including EF1α, EF1βγ, EF-Tu and EF-G;septins; tubulin; ARF related GTPase; rab; SSRP receptor; rhodopsin;transducin; and GTPase activating protein (GAP). Suitable phosphatasesinclude, but are not limited to, protein phosphatases; myosinphosphatase; IP3 phosphatase; pyrophosphatase; and Cdc25. Suitablephosphorylases include, but are not limited to, polynucleotidephosphorylase and glycogen phosphorylase.

By “ATPase” herein is meant an enzyme that hydrolyzes ATP. For example,ATPases include proteins comprising molecular motors such as kinesins,myosins and dyneins. “Molecular motor” is a molecule that utilizeschemical energy to produce mechanical force or movement; molecularmotors are particularly of interest in cytoskeletal systems. For furtherreview, see, Vale and Kreis, 1993, GUIDEBOOK TO THE CYTOSKELETAL ANDMOTOR PROTEINS New York: Oxford University Press; Goldstein, 1993, Ann.Rev. Genetics 27: 319–351; Mooseker and Cheney, 1995, Annu. Rev. CellBiol. 11: 633–675; Burridge et al., 1996, Ann. Rev. Cell Dev. Biol. 12:463–519.

In one embodiment, the target protein indirectly produces ADP orphosphate. In one aspect, a target protein indirectly produces ADP orphosphate by producing a product that then serves as a substrate in asubsequent enzymatic reaction for producing ADP or phosphate. Forexample, in a preferred embodiment, the target protein can be apyrophosphate producing enzyme. Suitable pyrophosphate producing enzymesinclude, but are not limited to, DNA polymerases; RNA polymerases;reverse transcriptase; DNA ligase; adenylate cyclase; guanylate cyclase;PRPP synthetase; TRNA synthetases; acyl CoA synthetase and acetyl CoAcarboxylase. Similarly, some ATPases produce AMP that can then be usedto make ADP.

In another embodiment, the target protein is a synthase. Thus, preferredsubstrates for producing phosphate include pyrophosphate and any of themono-, di- and triphosphate versions of CTP, UTP, GTP, ATP, and TTP, aswell as derivatives including dideoxy derivatives. Additionally, othersources of substrates that can be cleaved to phosphate includephosphorylated peptides, oligonucleotides, carbohydrates, lipids, etc.For example, inositol triphosphate (IP3) is an important signalingmoiety. Accordingly, any target protein which produces these compoundsor others that can be used to produce phosphate or ADP may be assayedusing the methods of the present invention.

In another aspect, a target protein indirectly produces ADP or phosphateby regulating an enzyme which produces phosphate or ADP. For example,the target can be an activator of an ATPase, such as an actin filamentor a microtubule; thus in this embodiment, the target protein may be aprotein polymer or oligomer. Alternatively, the target protein can be afilament binding protein or regulatory protein. For example, theregulatory protein can be the troponin-tropomyosin complex whichregulates the binding of myosin to actin. Since myosin's ATPase isactivated by binding to actin, modulators of this regulatory proteincomplex can be identified by the methods provided herein.

In a preferred embodiment, the target protein may consume ADP orphosphate; that is, rather than looking for an increase in signal, aloss of signal may be monitored.

Also included within the definition of the target proteins of thepresent invention are amino acid sequence variants of wild-type targetproteins. These variants fall into one or more of three classes:substitutional, insertional or deletional variants. As for the targetproteins as discussed below, these variants ordinarily are prepared bysite specific mutagenesis of nucleotides in the DNA encoding the targetprotein, using cassette or PCR mutagenesis or other techniques wellknown in the art, to produce DNA encoding the variant, and thereafterexpressing the DNA in recombinant cell culture as outlined above.However, variant target protein fragments having up to about 100–150residues may be prepared by in vitro synthesis using establishedtechniques. Amino acid sequence variants are characterized by thepredetermined nature of the variation, a feature that sets them apartfrom naturally occurring allelic or interspecies variation of the targetprotein amino acid sequence. The variants typically exhibit the samequalitative biological activity as the naturally occurring analogue,although variants can also be selected which have modifiedcharacteristics as will be more fully outlined below.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed variants screened for theoptimal combination of desired activity. Techniques for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known, for example, M13 primer mutagenesis and PCRmutagenesis. Screening of the mutants is done using assays of targetprotein activities.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to 20 amino acids, althoughconsiderably larger insertions may be tolerated. Deletions range fromabout 1 to about 20 residues, although in some cases deletions may bemuch larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative. Generally these changes are doneon a few amino acids to minimize the alteration of the molecule.However, larger changes may be tolerated in certain circumstances. Whensmall alterations in the characteristics of the target protein aredesired, substitutions are generally made in accordance with thefollowing chart:

CHART I Original Residue Exemplary Substitutions Ala Ser Arg Lys AsnGln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu,Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr SerThr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inChart I. For example, substitutions may be made which more significantlyaffect: the structure of the polypeptide backbone in the area of thealteration, for example the alpha-helical or beta-sheet structure; thecharge or hydrophobicity of the molecule at the target site; or the bulkof the side chain. The substitutions which in general are expected toproduce the greatest changes in the polypeptide's properties are thosein which (a) a hydrophilic residue, e.g. seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by)an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g. phenylalanine, is substituted for (orby) one not having a side chain, e.g. glycine.

The variants typically exhibit the same qualitative biological activity,although variants also are selected to modify the characteristics of thetarget proteins as needed. Alternatively, the variant may be designedsuch that the biological activity of the target protein is altered. Forexample, glycosylation sites may be altered or removed.

Further included within the definition of the target proteins of theinvention are covalent modifications of the target proteins. One type ofcovalent modification includes reacting targeted amino acid residues ofa target protein with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues of atarget protein. Derivatization with bifunctional agents is useful, forinstance, for crosslinking the target protein to a water-insolublesupport matrix or surface. Commonly used crosslinking agents include,e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxy-succinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79–86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the target proteins includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in the target native sequence,and/or adding one or more glycosylation sites that are not present inthe native sequence.

Addition of glycosylation sites to target polypeptides may beaccomplished by altering the amino acid sequence thereof. The alterationmay be made, for example, by the addition of, or substitution by, one ormore serine or threonine residues to the native sequence (for O-linkedglycosylation sites). The target amino acid sequence may optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the target polypeptide at preselected bases such thatcodons are generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on thetarget polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. Such methods are described in the art, e.g., in WO87/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259–306 (1981).

Removal of carbohydrate moieties present on the targget polypeptide maybe accomplished chemically or enzymatically or by mutationalsubstitution of codons encoding for amino acid residues that serve astargets for glycosylation. Chemical deglycosylation techniques are knownin the art and described, for instance, by Hakimuddin, et al., Arch.Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem.,118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

Another type of covalent modification of target proteins compriseslinking the target polypeptide to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol, polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

Target polypeptides of the present invention may also be modified in away to form chimeric molecules comprising a target protein fused toanother, heterologous polypeptide or amino acid sequence, a preferredembodiment of which is is described more fully below. In one embodiment,such a chimeric molecule comprises a fusion of a target polypeptide witha tag polypeptide which provides an epitope to which an anti-tagantibody can selectively bind. The epitope tag is generally placed atthe amino- or carboxyl-terminus of the target polypeptide. The presenceof such epitope-tagged forms of a target polypeptide can be detectedusing an antibody against the tag polypeptide. Also, provision of theepitope tag enables the target polypeptide to be readily purified byaffinity purification using an anti-tag antibody or another type ofaffinity matrix that binds to the epitope tag.

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159–2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610–3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering,3(6):547–553(1990)]. Other tag polypeptides include the Flag-peptide[Hopp et al., BioTechnology, 16:1204–1210 (1988)]; the KT3 epitopepeptide [Martin et al., Science, 255:192–194 (1992)]; tubulin epitopepeptide [Skinner et al., J. Biol. Chem., 266:15163–15166 (1991)]; andthe T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl.Acad. Sci. USA, 87:6393–6397 (1990)].

As will be appreciated by those in the art, the target proteins can bemade in a variety of ways, including both synthesis de novo and byexpressing a nucleic acid encoding the target protein.

Numerous suitable methods for recombinant protein expression, includinggeneration of expression vectors, generation of fusion proteins,introducing expression vectors into host cells, protein expression inhost cells, and purification methods are known to those in the art andare described, for example, in the following textbooks: Sambrook et al.,Molecular Cloning: A Laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989), Ausubel et al., Short Protocols in MolecularBiology (John Wiley & Sons, Inc., 1995), Harlow and Lane, Antibodies: ALaboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1988),O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual(New York: Oxford University Press, 1994), Richardson, BaculovirusExpression Protocols (Totowa: Humana Press, 1995), Kriegler, GeneTransfer and Expression: A Laboratory Manual (New York: OxfordUniversity Press, 1991), Roth, Protein Expression in Animal Cells,Methods in Cell Biology Vol. 43 (San Diego: Academic Press, 1994),Murray, Gene Transfer and Expression Protocols, Methods in MolecularBiology, Vol. 7 (Clifton: Humana Press, 1991), Deutscher, Guide toProtein Purification, Methods in Enzymology Vol. 182 (San Diego:Academic Press, Inc., 1990), Harris and Angal, Protein PurificationMethods: A Practical Approach (Oxford: IRL Press at Oxford UniversityPress, 1994), Harris and Angal, Protein Purification Applications: APractical Approach (Oxford: IRL Press at Oxford University Press, 1990),Rees et al., Protein Engineering: A Practical Approach (Oxford: IRLPress at Oxford University Press, 1992) and White, PCR Protocols,Methods in Molecular Biology, Vol. 15 (Totowa, Humana Press, 1993).

The selection of host cell types for the expression of target proteinswill depend on the target protein, with both eukaryotic and procaryoticcells finding use in the invention. Appropriate host cells includeyeast, bacteria, archebacteria, fungi, plant, insect and animal cells,including mammalian cells. Of particular interest are Drosophilamelangaster cells, Saccharomyces cerevisiae and other yeasts, E. coli,Bacillus subtilis, SF9 cells (and other related cells for use withbaculoviral expression systems), C 129 cells, 293 cells, Neurospora,BHK, CHO, COS, Dictyostelium, etc.

In a preferred embodiment, the target proteins are purified for use inthe assays, as outlined herein, to provide substantially pure samples.By “substantially pure” or “isolated” herein is meant that the proteinis unaccompanied by at least some of the material with which it isnormally associated in its natural state, preferably constituting atleast about 0.5%, more preferably at least about 5% by weight of thetotal protein in a given sample. A substantially pure protein comprisesat least about 75% by weight of the total protein, with at least about80% being preferred, and at least about 90% being particularlypreferred. Alternatively, the target protein need not be substantiallypure as long as the sample comprising the target protein issubstantially free of other components that can contribute to theproduction of ADP or phosphate (or, in the case of indirect assays,other components which are subsequently assayed).

The target proteins may be isolated or purified in a variety of waysknown to those skilled in the art depending on what other components arepresent in the sample. Standard purification methods includeelectrophoretic, molecular, immunological and chromatographictechniques, including ion exchange, hydrophobic, affinity, andreverse-phase HPLC chromatography, and chromatofocusing. For example,the target protein may be purified using a standard anti-target antibodycolumn. Ultrafiltration and diafiltration techniques, in conjunctionwith protein concentration, are also useful. For general guidance insuitable purification techniques, see Scopes, R., Protein Purification,Springer-Verlag, N.Y. (1982).

Suitable purification schemes for some specific kinesins are outlined inU.S. Ser. No. 09/295,612, filed Apr. 20, 1999, hereby expresslyincorporated herein in its entirety, along with referenced materials.

The present invention provides methods for screening for modulators oftarget proteins. By “modulators” herein is meant both antagonists andagonists of the target protein. Thus, “modulating the activity of thetarget protein” includes an increase in target protein activity, adecrease in target protein activity, or a change in the type or kind ofactivity present. Generally, the modulator will both bind to the targetprotein (although this may not be necessary), and alter its biologicalor biochemical activity as defined herein. For inhibitors, changes of25%, 50%, 75% and most preferably 100% of at least one biologicalactivity of the target protein is seen. For activators, preferably thechange is a change of at least 40%, more preferably at least 60%, morepreferably at least 80%, more preferably at least 100%, more preferablyat least 200%, and most preferably by at least 500%.

Accordingly, the present invention provides methods for screeningcandidate bioactive agents for the ability to modulate a targetprotein's activity. By “candidate agent” or “candidate bioactive agent”or “drug candidate” or grammatical equivalents herein is meant anymolecule, e.g., protein, oligopeptide, small organic molecule,polysaccharide, polynucleotide to be tested in a screening assay.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines (including derivatives, structural analogs, orcombinations thereof), derivatives, structural analogs or combinationsthereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification to producestructural analogs.

In an embodiment provided herein, the candidate bioactive agents areproteins. The protein may be made up of naturally occurring amino acidsand peptide bonds, or synthetic peptidomimetic structures. For example,homo-phenylalanine, citrulline and noreleucine are considered aminoacids for the purposes of the invention. “Amino acid” also includesimino acid residues such as proline and hydroxyproline. The side chainsmay be in either the (R) or the (S) configuration. In the preferredembodiment, the amino acids are in the (S) or L-configuration. Ifnon-naturally occurring side chains are used, non-amino acidsubstituents may be used, for example to prevent or retard in vivodegradations.

In another embodiment, the candidate bioactive agents are naturallyoccurring proteins or fragments of naturally occurring proteins. Thus,for example, cellular extracts containing proteins, or random ordirected digests of proteinaceous cellular extracts, may be used. In oneembodiment, the libraries are of bacterial, fungal, viral, and mammalianproteins, with the latter being preferred, and human proteins beingespecially preferred.

In one embodiment, the candidate agents are peptides of from about 2 toabout 30 amino acids, with from about 5 to about 20 amino acids beingpreferred, and from about 7 to about 15 being particularly preferred.The peptides may be digests of naturally occurring proteins as isoutlined above, random peptides, or random peptides. By randomized orgrammatical equivalents herein is meant that each nucleic acid andpeptide consists of essentially random nucleotides and amino acids,respectively. Since generally these random peptides (or nucleic acids,discussed below) are chemically synthesized, they may incorporate anynucleotide or amino acid at any position. The synthetic process can bedesigned to generate randomized proteins or nucleic acids, to allow theformation of all or most of the possible combinations over the length ofthe sequence, thus forming a library of randomized candidate bioactiveproteinaceous agents.

In one embodiment, the library is fully randomized, with no sequencepreferences or constants at any position. In a preferred embodiment, thelibrary is biased. That is, some positions within the sequence areeither held constant, or are selected from a limited number ofpossibilities. For example, in a preferred embodiment, the nucleotidesor amino acid residues are randomized within a defined class, forexample, of hydrophobic amino acids, hydrophilic residues, stericallybiased (either small or large) residues, towards the creation ofcysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., orto purines, etc.

In another embodiment, the candidate agents are nucleic acids. Bynucleic acid or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds, although in some cases, as outlined below, nucleic acid analogsare included that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl etal., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res.14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al.,J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (seeEckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press), and peptide nucleic acid backbones and linkages (seeEgholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed.Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al.,Nature 380:207 (1996), all of which are incorporated by reference).Other analog nucleic acids include those with positive backbones (Denpcyet al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones(U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863;Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991);Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et al.,Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASC SymposiumSeries 580, Carbohydrate Modifications in Antisense Research, Ed. Y. S.Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem.Lett. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994);Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, includingthose described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters6 and 7, ASC Symposium Series 580, Carbohydrate Modifications inAntisense Research, Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acidscontaining one or more carbocyclic sugars are also included within thedefinition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995)pp169–176). Several nucleic acid analogs are described in Rawls, C & ENews Jun. 2, 1997 page 35. All of these references are hereby expresslyincorporated by reference. These modifications of the ribose-phosphatebackbone may be done to facilitate the addition of additional moietiessuch as labels, or to increase the stability and half-life of suchmolecules in physiological environments.

In addition, mixtures of naturally occurring nucleic acids and analogscan be made. Alternatively, mixtures of different nucleic acid analogs,and mixtures of naturally occurring nucleic acids and analogs may bemade. The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xanthine,hypoxanthine, isocytosine, isoguanine, etc.

As described above generally for proteins, nucleic acid candidate agentsmay be naturally occurring nucleic acids, random nucleic acids, orbiased random nucleic acids. For example, digests of procaryotic oreukaryotic genomes may be used as is outlined above for proteins.

In a preferred embodiment, the candidate bioactive agents are organicchemical moieties, a wide variety of which are available in theliterature.

In a preferred embodiment, the candidate agent is a small molecule. Thesmall molecule is preferably 4 kilodaltons (kd) or less. In anotherembodiment, the compound is less than 3 kd, 2 kd or 1 kd. In anotherembodiment the compound is less than 800 daltons (D), 500 D, 300 D or200 D. Alternatively, the small molecule is about 75 D to 100 D, oralternatively, 100 D to about 200 D.

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 NWS, Advanced Chem Tech, LouisvilleKy.; Symphony, Rainin, Woburn, Mass.; 433A Applied Biosystems, FosterCity, Calif.; 9050 Plus, Millipore, Bedford, Mass.).

The present invention provides methods of screening candidate bioactiveagents for modulators of target protein activity. In a preferredembodiment, the methods are in vitro methods, utilizing purified orpartially purified target proteins. Alternatively, the methods are invivo methods, utilizing cells comprising target nucleic acids that canbe expressed to produce target proteins, particularly when the targetprotein is either secreted or on the surface.

In a preferred embodiment, the methods comprise combining a targetprotein and a candidate bioactive agent, and evaluating the effect onthe target protein's activity. By “target protein activity” orgrammatical equivalents herein is meant the biological activity of thetarget protein. As will be appreciated by those in the art, the activityof the target protein will vary with the target protein chosen, and willbe generally ascertainable by one of skill in the art of the targetprotein.

In a preferred embodiment, the methods of the invention comprise theaddition of candidate agents to the target proteins. In general, this isdone under conditions which normally allow the direct or indirectproduction of ADP or phosphate by the target protein. The phrase “underconditions which normally allow production or utilization of ADP orphosphate” as used herein means that all of the compositions andconditions are provided to allow the production or utilization of ADP orphosphate. Thus, the reaction which directly or indirectly produces oruses ADP or phosphate would normally occur in the absence of themodulator.

As will be appreciated by those in the art, the components are added inbuffers and reagents to assay target protein activity and give optimalsignals (i.e. the largest ADP or phosphate signals possible). Since themethods outlined herein allow kinetic measurements, the incubationperiods are optimized to give adequate detection signals over thebackground.

A “modulator of a target protein which directly or indirectly producesor uses ADP or phosphate” can be any compound as described herein in thecontext of candidate agents which modulates the target protein's director indirect production or use of ADP or phosphate relative to a control.

In one aspect, the method comprises subjecting the mixture to anenzymatic reaction which uses ADP or phosphate as a substrate underconditions which normally allow the ADP or phosphate to be utilized anddetermining the level of activity of the enzymatic reaction. This stepcan be performed in conjunction with identifying a modulator of a targetprotein which directly or indirectly produces ADP or phosphate orindependently thereof to identify a modulator of a protein which usesADP or phosphate.

The phrase to “use ADP or phosphate” as used herein means that the ADPor phosphate are directly acted upon. In one case, the ADP, for example,can be hydrolyzed or can be phosphorylated. As another example, thephosphate can be added to another compound. As used herein, in each ofthese cases, ADP or phosphate is acting as a substrate.

There are a number of enzymatic reactions known in the art which use ADPas a substrate. For example, kinase reactions such as pyruvate kinasesare well known. Nature, 78:632 (1956); Mol. Pharmacol, 6(1):31–40(1970). This is a preferred method in that it allows the regeneration ofATP. In one embodiment, the level of activity of the enzymatic reactionis determined directly. For example, in a pyruvate kinase reaction,pyruvate or ATP can be measured by conventional methods known in theart.

In a preferred embodiment, the level of activity of the enzymaticreaction which uses ADP as a substrate is measured indirectly by beingcoupled to another reaction. For example, in one embodiment, the methodfurther comprises a lactate dehydrogenase reaction under conditionswhich normally allow the oxidation of NADH, wherein said lactatedehydrogenase reaction is dependent on the pyruvate kinase reaction.Measurement of enzymatic reactions by coupling is known in the art,i.e., Nature, 178:632 (1956) and is further discussed below in regardsto fluorescence.

Furthermore, there are a number of reactions which utilize phosphate.Examples of such reactions include a purine nucleoside phosphorylasereaction. This reaction can be measured directly or indirectly. Thereaction can be measured directly by conventional methods known in theart.

In a preferred embodiment, the level of activity of the enzymaticreaction which uses phosphate as a substrate is measured indirectly bybeing coupled to another reaction. For example, in one embodiment, themethod further comprises a purine analog cleavage reaction underconditions which normally allow the cleavage of the purine analog. See,PNAS, 89:4884–4887 (1992); Anal. Biochem., 246:86–95 (1997); Biochem.,J., 266:611–614 (1990). Alternatively, xanthine oxidase can be used inconjunction with purine nucleoside phosphorylase to couple phosphateproduction to a change in the absorbance of a substrate for xanthineoxidase. Clin. Chim. Acta., 223:149–157 (1993).

In one embodiment, the detection of the ADP or phosphate proceedsnon-enzymatically, for example by binding or reacting the ADP orphosphate with a detectable compound. For example, phosphomolybdatebased assays may be used which involve conversion of free phosphate to aphosphomolybdate complex. J. Biol. Chem., 66:375–400 (1925). One methodof quantifying the phosphomolybdate is with malchite green. Chin. Chim.Acta, 14:361–366 (1966). Alternatively, a fluorescently labeled form ofa phosphate binding protein, such as the E. coli phosphate bindingprotein, can be used to measure phosphate by a shift in itsfluorescence.

In a preferred embodiment, detection of the assay is done using adetectable label. By “labeled” herein is meant that a compound has atleast one element, isotope or chemical compound attached to enable thedetection of the compound. In general, labels fall into three classes:a) isotopic labels, which may be radioactive or heavy isotopes; b)magnetic, electrical, thermal; and c) colored or luminescent dyes;although labels include enzymes and particles such as magnetic particlesas well. The dyes may be chromophores or phosphors but are preferablyfluorescent dyes, which due to their strong signals provide a goodsignal-to-noise ratio for detection. Suitable dyes for use in theinvention include, but are not limited to, fluorescent lanthanidecomplexes, including those of Europium and Terbium, fluorescein,rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin,methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow,Cascade Blue™, Texas Red, and derivatives thereof, and others describedin the 6th Edition of the Molecular Probes Handbook by Richard P.Haugland, hereby expressly incorporated by reference.

The invention provides methods of screening candidate agents for theability to serve as modulators of target protein activity. In apreferred embodiment, high throughput screening (HTS) systems are used,which can include the use of robotic systems. The assays of the presentinvention offer the advantage that many samples can be processed in ashort period of time. For example, plates having 96 or as many wells asare commercially available can be used.

High throughput screening systems are commercially available (see, e.g.,Zymark Corp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio;Beckman Instruments, Inc., Fullerton, Calif.; Precision Systems, Inc.,Natick, Mass., etc.) These systems typically automate entire proceduresincluding all sample and reagent pipetting, liquid dispensing, timedincubations, and final readings of the microplate in detector(s)appropriate for the assay. These configurable systems provide highthroughput and rapid start up as well as a high degree of flexibilityand customization. The manufacturers of such systems, i.e., ZymarkCorp., provide detailed protocols for the various high throughputassays.

Generally a plurality of assay mixtures are run in parallel withdifferent agent concentrations to obtain a differential response to thevarious concentrations. Typically, one of these concentrations serves asa negative control, i.e., at zero concentration or below the level ofdetection. However, in one embodiment, any concentration can be used asthe control for comparative purposes.

In one preferred embodiment, high throughput screening methods involveproviding a library containing a large number of compounds (candidatecompounds) potentially having the desired activity. Such “combinatorialchemical libraries” are then screened in one or more assays, asdescribed herein, to identify those library members (particular chemicalspecies or subclasses) that display a desired characteristic activity.The compounds thus identified can serve as conventional “lead compounds”or can themselves be used as potential or actual therapeutics oragricultural compounds.

For example, in one embodiment, candidate agents are assayed in highlyparallel fashion by using multiwell plates by placing the candidateagents either individually in wells or testing them in mixtures. Assaycomponents, such as for example, molecular motors, protein filaments,coupling enzymes and substrates, and ATP can then be added to the wellsand the absorbance or fluorescence of each well of the plate can bemeasured by a plate reader. A candidate agent which modulates thefunction of the molecular motor is identified by an increase or decreasein the rate of ATP hydroylsis compared to a control assay in the absenceof that candidate agent.

A preferred HTS system is as follows. The system comprises a microplateinput function which has a storage capacity matching a logical “batch”size determined by reagent consumption rates. The input device storesand, delivers on command, barcoded assay plates containing pre-dispensedsamples, to a barcode reader positioned for convenient and rapidrecording of the identifying barcode. The plates are stored in asequential nested stack for maximizing storage density and capacity. Theinput device can be adjusted by computer control for varying platedimensions. Following plate barcode reading, the input device can beadjusted by computer control for varying plate dimensions. Followingplate barcode reading, the input device transports the plate into thepipetting device which contains the necessary reagents for the assay.Reagents are delivered to the assay plate with the pipetting device. Tipwashing in between different reagents is performed to prevent carryover.A time dependent mixing procedure is performed after each reagent toeffect a homogeneous solution of sample and reagents. The sequentialaddition of the reagents is delayed by an appropriate time to maximizereaction kinetics and readout levels. Immediately following the lastreagent addition, a robotic manipulator transfers the assay plate intoan optical interrogation device which records one or a series ofmeasurements to yield a result which can be correlated to an activityassociated with the assay. The timing of the robotic transfer isoptimized by minimizing the delay between “last reagent” delivery andtransfer to the optical interrogation device. Following the opticalinterrogation, the robotic manipulator removes the finished assay platesto a waste area and proceeds to transfer the next plate from pipettingdevice to optical interrogation device. Overlapping procedures of theinput device, pipetting device and optical interrogation device are usedto maximize throughput.

It is understood that the methods provided herein can be applied to avaried array of target proteins and are not limited to cytoskeletalcomponent systems. However, for illustrative purposes, another exampleof the present invention is to assay for modulators of the polymerizedstate of cytoskeletal filament proteins actin or tubulin. In thisexample, the candidate agent or mixture comprising at least onecandidate agent is incubated with the filament protein under conditionsthat would normally promote either polymerization or depolymerization. Amolecular motor that is activated by the filament is then added to theassay mixture and its activity is monitored by ADP or phosphate releaseas discussed above. Candidate agents which increase the fraction of thefilament protein in a polymerized state will be identified by anincrease in the motor ATPase and those which increase the fraction ofthe filament protein in a depolymerized state will be identified by adecrease in the motor ATPase.

It is understood that once a modulator or binding agent is identifiedthat it can be subjected to further assays to further confirm itsactivity. In particular, the identified agents can be entered into acomputer system as lead compounds and compared to others which may havethe same activity. The agents may also be subjected to in vitro andpreferably in vivo assays to confirm their use in medicine as atherapeutic or diagnostic or in the agricultural arena.

In a preferred embodiment, approximately 1000 assays are performed perhour with very low false negative and false positive rates, with up to10,000 assays an hour being preferred and more than 10,000 assays perhour being particularly preferred. In a particularly preferredembodiment, at least one or more of the steps regarding automated liquidhandling or preferred assay design as described herein are included.

In one embodiment, the method comprises automated liquid handling.

In preferred embodiment, an antifoam or a surfactant is included in theassay mixture and wash solution. Suitable antifoams include, but are notlimited to, antifoam 289 (Sigma), and others commercially available.Suitable surfactants include, but are not limited to, Tween, Tritonsincluding Triton X-100, saponins, and polyoxyethylene ethers. Thiseliminates bubbles which often result in conventional methods requiringpipetting into low volume assay wells. Thus, in a preferred embodiment,the invention includes the use of an antifoam, detergent or surfactantas a reagent in a high throughput screens, including, but not limited tothe screens of the invention. Generally the antifoams, detergents orsurfactants are added at a range from about 0.01 ppm to about 10 ppm,with from about 1 to about 2 ppm being preferred. In a further preferredembodiment, the invention includes the use of an antifoam, surfactant ordetergent when the assay requires mixing, particularly physical mixingsuch as shaking the microtiter plates. In an additional preferredembodiment, the invention includes the use of an antifoam, surfactant ordetergent when the assay is done in microtiter plates, particularlyplates with 96 wells or more, including 96, 384 and 1536 plates.

In another aspect, a round sample well is used. This helps increase thepathlength for absorbance measurements for a given assay volume andhelps flatten the meniscus of the solution in each assay well.Preferably, the method comprises vigorous shaking of the sample platefollowing the addition of each reagent.

In a preferred embodiment herein, a preferred assay design is provided.In one aspect, the preferred assay preferably uses a multi-time-point(kinetic) assay, with at least two data points being preferred. As willbe appreciated by those in the art, the interval can be adjusted tocorrelate with the biological activity of the protein. In the case ofmultiple measurements the absolute rate of the protein activity can bedetermined, and such measurements have higher specificity particularlyin the presence of candidate agents which have similar absorbance orfluorescence properties to that of the enzymatic readout. The kineticassay reduces the false positive rate. In an additional aspect, thekinetic rate are normalized to several control wells on each assayplate. This allows for some variation in the activity of the targetproteins and the stability of assay reagents over time and thus permitsscreening runs of several hours.

When proteins that use ATP are included, the pyruvate kinase/lactatedehydrogenase embodiments are particularly preferred due to theadvantage of ATP regeneration so that ATP concentration is constant overtime.

Further regarding variation of the assays, it is understood that for akinesin-microtubule modulator assay, the order of addition of the assaycomponents affects the ATPase rate.

The invention further provides methods for identifying whether any twotest proteins interact. Briefly, the assay is functionally similar to ayeast two-hybrid system, but relies on an increase in ATPase activity asa result of bringing two components together as a result of aprotein—protein interaction. As an example, the system is describedusing a biological polymer binding site and a polymer stimulated ATPase,although as will be appreciated by those in the art, any two componentsthat result in an increase in ATPase activity as a result of associationcan be used. For example, a first test protein (a “bait” protein), forwhich an interaction is sought, is joined, usually covalently, to abiological polymer binding protein, for example a cytoskeletal bindingprotein (such as a microtubule binding protein) to form a first targetchimera. The term “chimera” or “fusion protein” as used herein refers toa protein (polypeptide) composed of two polypeptides that, whiletypically unjoined in their native state, typically are joined by theirrespective amino and carboxyl termini through a peptide linkage to forma single continuous polypeptide. It will be appreciated that the twopolypeptide components can be directly joined or joined through apeptide linker/spacer.

A second test protein (a “prey” protein), is joined, again usuallycovalently, to an ATPase domain that is stimulated by the cytoskeletalcomponent to form a second target chimera. Upon combination with thecytoskeletal component, the first target chimera binds to thecytoskeletal component, and if the first and second target proteinsinteract, the second target chimera is brought into proximity with thecytoskeletal component, and thus the ATPase activity is stimulated andcan be detected. If there is no interaction, no increase in ATPproduction is observed.

In a preferred embodiment, the biological polymer binding proteincomprises just a domain of a larger protein that comprises an ATPasedomain; that is, the ATPase domain has been removed. Alternatively, thebiological polymer binding protein may include the larger protein buthave the ATPase domain inactivated, for example by mutation. Similarly,the ATPase domain may be either just the ATPase functional domain of aprotein, or it may include a larger protein that has the binding domaininactivated.

As discussed above, the chimera proteins are generally joinedcovalently, for example by making fusion proteins, although covalentcross-linking can be used, or high affinity non-covalent associationscan also be done, for example using binding partners such asbiotin/avidin, etc. In a preferred embodiment, the fusion proteins aremade using fusion genes, as is generally known in the art.

In a preferred embodiment, the target proteins should not have ATPaseactivity themselves, although it is possible to detect increases inactivity.

Suitable biological polymers include, but are not limited to, nucleicacids including DNA and RNA, and cytoskeletal components including, butnot limited to, microtubules and microfilaments (actin filaments).

Suitable biological binding sites include, but are not limited to,nucleic acid binding domains (when nucleic acids are the biologicalpolymer), and molecular motor binding domains (in the case ofcytoskeletal components).

Suitable ATPases include, but are not limited to, those that exhibit anincrease (stimulation) in the presence of the biopolymer, such as DNAand RNA polymerases in the case of nucleic acids, microtubule stimulatedATPases in the case of microtubules including kinesins and dyneins, andactin stimulated ATPases such as myosins.

In a preferred embodiment, the first test protein is attached to afunctional molecular motor binding domain to provide a first targetchimera. The second test protein is attached to a functional microtubulestimulated ATPase domain to form a second target chimera. The first andsecond target chimeras are combined under conditions which normallyallow activity of a motor protein which comprises a molecular motorbinding domain and a microtubule stimulated ATPase domain. An increasein motor protein activity indicates interaction between the two testproteins.

Customarily one bait protein is used to test a library of test sequencesas is described below; however, as will be appreciated by those in theart, the bait protein may be one of a library as well, thus forming anexperimental matrix wherein two libraries (although the coding regionsof the libraries could be identical) are evaluated for protein—proteininteractions. In a preferred embodiment, self-activating bait proteinsare filtered out from the bait protein library.

In another embodiment a method for identifying whether a candidate agentis a modulator of at least one of a first and second test protein isprovided. In this case, a candidate agent is combined with the first andsecond chimeras as described above. A change in molecular motor activityin the presence and absence of the candidate agent indicates that thecandidate agent is a modulator of at least one of the two candidateagents.

Thus, the chimeras of the present invention can be formed usedrecombinant techniques known in the art. The chimera can be formed atthe protein level wherein two polypeptides are joined, or at themolecular level wherein a nucleic acid is formed which encodes theappropriate functional motor component and the appropriate test protein.

In a preferred embodiment, the nucleic acids encoding a chimera are usedto express the respective recombinant chimera. A variety of expressionvectors, including viral and non-viral expression vectors can be madewhich are useful for recombinant protein expression in a variety ofsystems, including, but not limited to, yeast, bacteria, archaebacteria,fungi, insect cells and animal cells, including mammalian cells.

The expressed chimera may also include further fusion domains includingtag polypeptides. Recombinant protein is produced by culturing a hostcell transformed with a nucleic acid encoding the chimera (generally asan expression vector), under the appropriate conditions that induce orcause expression of the chimera.

In a preferred embodiment, the recombinant chimera is purified followingexpression, as outlined above.

For using the chimeras in the assays described herein, if the two testproteins bind to one another, a complex with both chimeras comprising afunctional molecular motor is formed. Thus, the binding interactionbetween the two test proteins can be identified by functional motoractivity under conditions which would normally allow motor activity ifboth a functional microtubule stimulated ATPase and binding domain werepresent.

In the case of identifying a modulator in an assay utilizing thechimeras of the present invention, the modulator can be an activator ofthe motor activity. Thus, in the absence of the candidate agent, theremay be no motor activity, however, in the presence of the candidateagent, motor activity occurs. Conversely, there may be significant motoractivity, indicating that the two testbinding proteins interact, butthis may decrease in the presence of a candidate agent. In either case,the candidate agent is identified as a modulator of at least one the twotest proteins.

In a preferred embodiment, motor activity is identified by ATPhydrolysis as described above. However, it is understood that motoractivity can be identified by a number of assays. Such assays includemicrotubule gliding, depolymerization/polymerization and any motoractivity which requires both binding and ATPase activity. Therefore, inthe case that the molecular motor used has another specific activity,such as involvement in mitosis or axonal transport, specific assays forthose activities can be utilized.

Generally motility assays involve immobilizing one component of thesystem (e.g., the kinesin motor or the microtubule) and then detectingmovement, or change thereof, of the other component. Thus, for example,in a preferred embodiment, the microtubule will be immobilized (e.g.,attached to a solid substrate) and the movement of the kinesin motormolecule(s) will be visually detected. Typically the molecule that is tobe detected is labeled (e.g., with a fluorescent label) to facilitatedetection.

Methods of performing motility assays are well known to those of skillin the art (see, e.g., Hall, et al. (1996), Biophys. J, 71: 3467–3476,Turner et al., 1996, Anal. Biochem. 242 (1):20–5; Gittes et al., 1996,Biophys. J. 70(1): 418–29; Shirakawa et al., 1995, J. Ex. Biol. 198:1809–15; Winkelmann et al., 1995, Biophys. J. 68: 2444–53; Winkelmann etal., 1995, Biophys. J. 68: 72S, and the like).

In addition to the assays described above for identifying ATPaseactivity, conventional methods can be used. For example, P_(i) releasefrom kinesin can be quantified. In one preferred embodiment, the ATPaseactivity assay utilizes 0.3 M PCA (perchloric acid) and malachite greenreagent (8.27 mM sodium molybdate II, 0.33 mM malachite green oxalate,and 0.8 mM Triton X-100). To perform the assay, 10 μL of reaction isquenched in 90 μL of cold 0.3 M PCA. Phosphate standards are used sodata can be converted to mM inorganic phosphate released. When allreactions and standards have been quenched in PCA, 100 μL of malachitegreen reagent is added to the to relevant wells in e.g., a microtiterplate. The mixture is developed for 10–15 minutes and the plate is readat an absorbance of 650 nm. If phosphate standards were used, absorbancereadings can be converted to mM P_(i) and plotted over time.

Additionally, in the case of methods provided herein utilizing thechimeras in accordance with the present invention, the remaining ATP canbe measured using the luciferin-luciferase system. Anal. Biochem.,40:1–17 (1971).

The assays are preferably performed in a high throughput system asdescribed herein utilizing multiwell plates and fluorescence orabsorbance readouts.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety.

EXAMPLE A High Throughput Assay for Modulators of the Molecular MotorKinesin

This assay is based on detection of ADP production from kinesin'smicrotubule stimulated ATPase. ADP production is monitored by a coupledenzyme system consisting of pyruvate kinase and lactate dehydrogenase.Under the assay conditions described below, pyruvate kinase catalyzesthe conversion of ADP and phosphoenol pyruvate to pyruvate and ATP.Lactate dehydrogenase then catalyzes the oxidation-reduction reaction ofpyruvate and NADH to lactate and NAD+. Thus, for each molecule of ADPproduced, one molecule of NADH is consumed. The amount of NADH in theassay solution is monitored by measuring light absorbance at awavelength of 340 nm.

Assay Components

A kinesin heavy chain construct consisting of the N-terminal 420 aminoacids is used in the assay. The final 25 μl assay solution consists ofthe following: 5 μg/ml kinesin, 30 μg/ml microrubules, 5 μM Taxol, 0.8mM NADH, 1.5 mM phosphoenol pyruvate, 3.5 U/ml pyruvate kinase, 5 U/mllactate dehydrogenase, 25 mM Pipes/KOH pH 6.8, 2 mM MgCl2, 1 mM EGTA, 1m MDTT, 0.1 mg/ml BSA, 0.001% antifoam 289 (Sigma), and 1 mM ATP.

Compound Plates

Potential chemical modulators of kinesin are dissolved in DMSO at aconcentration of approximately 1 mg/ml, and 0.5 μl of each chemicalsolution is dispensed into a single well of a clear 384 well plate(Clinipate, Labsystems). On each plate, there are at least 16 wells intowhich pure DMSO (without a candidate compound) is dispensed. These wellsserve as negative controls for comparison to the potential chemicalmodulators on that plate. The compound plates are made in advance andstored at 4° C., and each plate is labeled with a bar code which is usedto identify the compounds on a given plate.

Instrumentation

The robotic system that runs the assay consists of a plate storage andretrieval device (Plate Stak, CCS Packard), a 96 channel automatedpipetting device (Multimek, Beckman), a robotic arm (Twister, Zymark),and a plate reader for absorbance (Ultramark, BioRad). The system iscontrolled by a custom-built software application.

Assay Performance

A stack of compound plates is placed in the plate storage devices andplates are transferred one at a time to the automated pipetting deviceby the plate carrier of the Plat Stak. Each of the 384 wells are thenfilled with 20 μl of a solution consisting of all of the assaycomponents described above except for ATP. The plate is then agitated athigh frequency by rapidly moving the plate carrier between two positionsthat are separated by a few millimeters. The plate is then returned tothe pipetting position. While the shaking of the plate occurs, the pipettips are washed with a solution of 0.001% antifoam in deionized water.To start the assay, 5 μl of a second solution containing ATP is thenadded to each well. The solution is then mixed by a second cycle of highfrequency agitation. The plate is then transferred to the plate readerby the robotic arm. In the plate reader, 10 absorbance measurements at340 nm are taken at 12 second intervals to produce a 2 minute kineticread for each well. While one plate is being read, the next plate istransferred to the pipetting device and prepared up to but not includingthe addition of the second solution. When the plate read is complete,the robotic arm transfers the plate to a waste chute and simultaneouslythe second solution is pipetted into the next plate so that it can betransferred to the reader to complete the cycle. The entire assay is runat room temperature ˜20° C.

Data Analysis

Following data acquisition, the maximum rate of the absorbance change iscalculated for each well and normalized to the average of the controlwells (without compound) which were present on the same plate. Thenormalized rates are then entered into an Oracle database, and thisallows them to be correlated with the potential chemical modulators. Oneach plate, the coefficient of variation of the slopes for the controlwells ranges from 48%. Quality control is assured by monitoring for aminimal initial absorbance and a linear absorbance change.

Important Features

There are several features of this system which are important. Thekinetic design which consists of multiple absorbance measurementsdramatically improves the specificity of the assay over a singleendpoint measurement. First, the rate of the reaction is to a firstapproximation independent of small differences between wells in the timefrom the start of the reaction to the first reading, and as a result,the overall variation in the data is reduced. Second, the rate of theabsorbance change is not affected by having a chemical compound whichabsorbs light of the same wavelength.

The presence of control wells in each plate and the subsequentnormalization of the data to those wells allows data to be taken forseveral hours despite some degradation of the enzyme activities whichresults from the aging of the solutions. This also improves thereproducibility of the data.

The presence of antifoam in the solution and the tip washing solutionimproves overall liquid handling by reducing the number of trappedbubbles in the small wells and helps flatten the fluid meniscus in eachwell for more reliable absorbance measurements. Additional featureswhich improve liquid handling are the vigorous shaking of the platedescribed above, and the round shape of the wells in the microplatesused.

The assay components and the performance of the assay are optimizedtogether to match the overall read time with the rate of kinesin's ADPproduction. In this example, the rate of absorbance change isapproximately 150–250 mOD/min. This corresponds to the production ofapproximately 2 μM ADP/sec. In addition to optimizing the rate of ADPproduction, the read time must be long enough for the rate of NADHconsumption to reach steady state beyond an initial lag time of severalseconds. In some cases, the order of addition of the reagents can have asignificant affect on the rate of ADP production. In the above example,the optimal rate is achieved by premixing all reagents except for thecompound of interest and ATP.

1. A high throughput assay method of identifying a candidate agent as amodulator of the function of a target protein wherein said methodcomprises: a) adding a candidate agent to a mixture comprising a targetprotein which directly or indirectly produces ADP or phosphate underconditions which normally allow the production of ADP or phosphate; b)subjecting the mixture to an enzymatic reaction which uses said ADP orphosphate as a substrate under conditions which normally allow the ADPor phosphate to be utilized; and c) determining the level of activity ofthe enzymatic reaction wherein a change in said level between thepresence and absence of said candidate agent indicates that saidcandidate agent is a modulator of said target protein function, whereinat least 1,000 assays and up to about 10,000 assays can be performed perhour and said assays are performed in microtiter plates having 96 wellsor more.
 2. The method of claim 1, wherein the mixture further comprisesa surfactant or antifoam.
 3. The method of claim 1, wherein thedetermining occurs by a fluorescent, luminescent, radioactive orabsorbance readout.
 4. The method of claim 1, wherein the enzymaticreaction is determined at multiple time points.
 5. The method of claim1, wherein a plurality of candidate agents are added.
 6. The method ofclaim 1, wherein a plurality of candidate agents are added to aplurality of target proteins.
 7. The method of claim 1, wherein thecandidate agents are added using a robotic system.
 8. The method ofclaim 1, wherein the adding step a and the subjecting step b areperformed simultaneously.
 9. A high throughput assay method ofidentifying a candidate agent as a modulator of the function of a targetprotein wherein said method comprises: a) adding a candidate agent to amixture comprising a target protein which directly or indirectlyproduces ADP or phosphate under conditions which normally allow theproduction of ADP or phosphate; b) subjecting the mixture to anenzymatic reaction which uses said ADP or phosphate as a substrate underconditions which normally allow the ADP or phosphate to be utilizedwherein said enzymatic reaction comprises a purine nucleosidephosphorylase and a purine analog; and c) determining cleavage of thepurine analog in the presence and absence of said candidate agent as ameasure of phosphate production wherein a change in cleavage of thepurine analog between the presence and absence of said candidate agentindicates that said candidate agent is a modulator of said targetprotein function, wherein at least 1,000 assays and up to about 10,000assays can be performed per hour and the assays are performed inmicrotiter plates having 96 wells or more.
 10. The method of claim 9,wherein the mixture further comprises a surfactant or antifoam.
 11. Themethod of claim 9, wherein the determining occurs by a fluorescent,luminescent, radioactive or absorbance readout.
 12. The method of claim9, wherein the enzymatic reaction is determined at multiple time points.13. The method of claim 9, wherein a plurality of candidate agents areadded.
 14. The method of claim 9, wherein a plurality of candidateagents are added to a plurality of target proteins.
 15. The method ofclaim 9, wherein the candidate agents are added using a robotic system.16. The method of claim 9, wherein the adding step a and the subjectingstep b are performed simultaneously.